重组人促性腺激素释放激素及导肽的分离纯化与活性分析

Purification and Activity Analysis of Recombinant Human GnRH and Leader Peptide

对含重组人促性腺激素释放激素(GnRH)及导肽(LEP)的工程菌株进行诱导表达,分离纯化GnRH/LEP并进行生物学活性分析。工程菌IPTG诱导,收获的菌体经超声破碎后,裂解液用Glutathione-Sepharose 4B亲和层析纯化GST-GnRH/LEP融合蛋白,经CNBr裂解、Sephadex G-25柱脱盐、QAE-Sepharose FF阴离子交换柱层析和RP-C18柱脱盐,得到纯度大于98%的重组GnRH/LEP。Western blot表明表达产物均具有GnRH抗原特异性。生物学活性分析表明:该表达产物可促进小鼠次级卵泡向成熟卵泡发育,可提高其血清中E2含量。

To purify recombinant Human GnRH and leader pepdde （GnRH/LEP）and study its biological acdvity from fusion protein GST-GnRH/LEP expressed in E.coli BL21 （DE3）PpGEX-6p-1-GnRH/LEP. E.coli BL21 （DE3） PpGEX-6p-1-GnRH/LEP were abducted by IPTG,GST-GnRH/LEP fusion protein was prepared from sonicadon of bacteria by glutathione-Sepharose 4B affinity chromatography. Following cleavage of GST-GnRH/LEP by cyanogen bromide,the recombinant GnRH/LEP was released from fusion protein,and purified using Sephadex G-25,QAE- Sepharose FF and RP-C18 column chromatography. The purity of recombinant GnRH/LEP was more than 98% by HPLC analysis. After purification,Western blot proved GnRH antigenic specificity of expressed product.The biological activity was examined by intra peritoneal injecdon of GnRH/LEP into mice,as compared with H2O-injecdon control group,The recombinant GnRH/LEP sdmulared the growth of mouse＇s follicle.And stimulated the secondary follicle to mature follicle It raised the content of E2 in mouse＇s serum,but it had different effect on different body.